RNA干涉（RNAi）技术应用于哺乳动物细胞的研究策略

RNAi作为新近发展起来的基因功能分析技术,近年来在哺乳动物细胞中的研究已取得了长足进展,且有着广泛的应用前景。对RNAi作用机制及RNAi实验操作技术的探讨是目前研究的热点。研究表明,哺乳动物细胞中的RNAi作用模式与植物有所不同。文章对RNAi作用机制、哺乳动物细胞RNAi实验的一般策略（包括靶siRNA序列选择、siRNA获取方法、siRNA转染、RNAi效果检测等）以及最新研究进展进行简述,以供类似工作的参考。     

基于BP神经网络的siRNA活性预测

RNA干扰(RNAinterference,RNAi)是由双链RNA(dsRNA)引起的基因沉默现象,它通过降解具有同源序列的mRNA来起作用,特殊设计的siRNA能使靶基因发生特异性沉默,起到确定基因功能或沉默致病基因从而治疗疾病的目的。在RNAi技术的应用中,通常采用的是长度为19bp,正、反义链3'端各有2个不配对碱基的双链RNA(siRNA)。但针对靶基因不同位点设计的siRNA作用效果差别很大。影响siRNA效果的因素是多方面的,这些因素的作用又是非线性的。本文在研究影响siRNA作用效果的各种因素的基础上,对已经公开发表的实验数据进行特征提取,作为BP神经网络的训练数据,并将训练好的BP神经网络用于siRNA活性预测。     

国外动态

siRNA检测芯片 :RNAi基因沉默新技术Genetix公司研制成功一系列适合检测RNA干扰研究中siRNA分子干扰效果的基因芯片产品。在即将于 2 0 0 4年 10月召开的RNAi欧洲会议上 ,Genetix将会介绍他们的QAr rayMini生物芯片和可选择蓝色激光的aQuire基因芯片扫描仪 ,这一组合是进行RNAi优化实验的完美配套产品。RNAi已经发展成一种强大的分子生物学研究工具 ,用于沉默细胞中的目标基因。研究人员能够使用各种不同的技术将双链siRNA分子 (一般长度为 2 0 2 5个核苷酸 )引入到哺乳动物细胞中。一旦进入细胞 ,这些分子就会与互补的目标mR…     

干扰RNA（RNAi）作用机理及其在植物细胞工程中的应用进展

随着RNAi调控目的基因表达机理的逐渐深入研究,RNAi技术也发展为一种强有力的实验工具,用来控制目的基因的表达以获取预期的生物表型。目前在植物中至少发现存在三种不同的RNAi途径,这些途径中基因沉默信号可以放大、传递和自我调控。为了建立高效、经济的RNAi技术体系,必须解决以下几个问题：即RNAi的有效传递,稳定性的提高,非目标效应出现的减小以及目标RNA敏感位点的确定等。本文综述了RNAi作用机制以及其在植物细胞工程中的最新应用进展,并详细探讨了其技术体系。     

基于绿色荧光蛋白的RNAi技术在细胞生物学实验课程中的应用实例

RNAi和GFP报告基因的转染技术是细胞生物学领域常用的研究手段,我们以GFP作为靶基因,设计了适合在本科生课程中开设的RNA干扰技术实验项目,本教改研究将GFP荧光观察、基因转染技术及RNA干扰技术融合在一个实验中,并且对该实验项目的实例应用做了细致的设计和具体实践,结果表明RNAi技术对细胞中GFP报告基因表达的抑制效果明显。此外本文还阐述了该实验项目在本科生的细胞生物学实验课程中开展的可行性和必要性。这种将传统技术与新技术交叉组合型实验项目的开展对于学生综合科研能力的培养具有良好的效果,也为细胞生物学实验课程的改革提供了新思路。     

UL29shRNA表达质粒与ACV对HSV-2 抑制效果的比较

目的:通过将筛选的UL29shRNA表达质粒与抗病毒药物阿昔洛韦(ACV)的抗病毒效果及细胞毒性进行比较,探讨RNA干扰在细胞水平上对HSV-2病毒的抑制效果。方法:将筛选的干扰效果最好的表达质粒UL29-shRNA1641作为RNA干扰组与抗病毒药ACV进行对HSV-2的抑制效果的比较研究,分为RNAi组、ACV组和RNAi+ACV组。采用实时荧光定量PCR检测UL29mRNA的相对表达量,Karber法检测细胞上清液中病毒滴度的变化、蛋白印迹法(Western blot)检测ICP8的相对表达量,对RNAi与ACV抑制效果进行比较,通过WST-1(Water-Soluble Tetrazolium-1)法对质粒转染复合物(质粒+转染试剂)和ACV的细胞毒性进行比较。结果:RT-PCR检测三组mRNA的相对表达水平,其中RNAi组UL29基因相对表达量高于ACV组(P0.05);RNAi+ACV组UL29基因相对表达量明显低于RNAi组和ACV组(P0.05)。Karber法检测三组细胞上清液病毒滴度表明,RNAi组病毒滴度高于ACV组(P0.05),RNAi+ACV组的病变病毒滴度明显低于RNAi组和ACV组(P0.05)。Western blot检测ICP8相对表达量,ACV组的ICP8(UL29编码的单链DNA结合蛋白,主要作用是在HSV-2 DNA复制过程中与复制叉产生了单链DNA结合,防止其重新配对形成ds DNA或被核酸酶降解)相对表达量比RNAi组低(P0.05)。RNAi+ACV组ICP8相对表达量降低最为明显,与ACV组和RNAi组相比有显著差异(P0.05)。WST-1法对转染复合物与ACV的细胞毒性进行比较,其中RNAi中质粒和转染试剂对细胞的毒性明显小于ACV。结论:在RNAi与ACV对HSV-2抑制效果的比较中,ACV要好于RNAi,两者联合使用的抑制效果好于单独使用RNAi或ACV。在抑制效果都较好的情况下比较,RNAi中使用的质粒与转染试剂对细胞的影响比ACV小。     

RNAi递送系统在害虫防治中的研究进展

RNAi是由dsRNA引发的,靶向目的 基因的高效与特异的基因沉默技术.由于其高效性、特异性和便捷性,RNAi技术广泛应用于基因功能研究、高通量目的 基因筛选、基因治疗、药物靶标预测和农业病虫害防治等领域.考虑到RNAi效率、安全性和预期靶基因下调的潜在障碍,RNAi提供高效防治应用的前提是以合适的方式递送效应RNA.本文对近期农业重要性害虫和病媒害虫防治应用中开发的RNAi递送系统进行综述,对这些RNAi递送系统的干扰效率和多重进入位点作了比较,并展望了RNAi递送系统在害虫防治中的应用,以期更好地完善RNAi技术在昆虫学研究和害虫防治中的应用.     

昆虫的RNA干扰

RNA干扰(RNAi)是一种强有力的分子生物学技术, 在昆虫研究中得到了较多的应用。目前, RNAi技术主要应用于昆虫功能基因和功能基因组研究, 已在多个目的19种昆虫上实现了RNAi。在昆虫上实现RNAi的方法主要有注射、浸泡、喂食、转基因和病毒介导等方法, 这些方法各有特点, 其中喂食法因其简单而最有应用前景。昆虫RNAi的系统性较为复杂, 只有部分昆虫具有RNAi的系统性。昆虫中RNAi信号传导的基因可能是sid-1, 但昆虫RNAi的系统性机理还不是很清楚。转基因植物产生的dsRNA实现了对作物的保护, 证实了RNAi技术可用于害虫控制, 为害虫控制开辟了新领域。昆虫的RNAi研究处在起步阶段, 研究昆虫RNAi的机理, 特别是RNAi在昆虫体内的系统性扩散机理, 改进实现RNAi的方法, 提高RNAi技术在昆虫研究中的应用, 有利于昆虫基因功能鉴定和害虫控制, 促进昆虫学科的发展。     

RNA干涉的临床前实验研究进展

RNA干涉（RNA interference,RNAi）是一种非常保守的细胞现象,在基础研究和疾病治疗方面具有重大的应用前景。尤其引人注目的是,新的RNAi方法学与已建立的转基因策略相结合,有效地将组织特异性RNAi导入到患者体内,有望治疗人类疾病。本文综述了RNAi的机制与在其临床前的实验研究,简要介绍了RNAi的新用途,讨论了RNAi基因治疗存在的问题,展望了RNAi基因治疗的应用前景。     

昆虫RNAi效率的影响因素研究进展

RNA干扰(RNAi)是一种在动植物中广泛存在的、由双链RNA诱发的、导致mRNA特异性沉默的过程.简单总结昆虫RNAi的研究现状,从理论和实践两个方面重点介绍昆虫RNAi效率影响因素的研究进展,最后对昆虫RNAi应用于害虫防治的前景进行展望.     

昆虫RNAi技术及其应用

RNAi是近几年发展起来的抑制基因表达的新技术。部分昆虫存在RNAi信号的系统性传播现象,可以将dsRNA直接注射进昆虫的卵、血腔或局部组织,引发远距离靶基因的特异性沉默,建立起了Embryo RNAi,Larval RNAi,Adult RNAi,Parental RNAi,Feeding RNAi和基于转基因技术的可遗传RNAi等昆虫RNAi技术,使RNAi迅速成为了研究昆虫尤其是非模式昆虫基因功能的主要方法。文章拟就RNAi的系统性、昆虫RNAi技术及其应用进行综述。     

Dissecting Systemic RNA Interference in the Red Flour Beetle Tribolium castaneum: Parameters Affecting the Efficiency of RNAi

The phenomenon of RNAi, in which the introduction of dsRNA into a cell triggers the destruction of the corresponding mRNA resulting in a gene silencing effect, is conserved across a wide array of plant and animal phyla. However, the mechanism by which the dsRNA enters a cell, allowing the RNAi effect to occur throughout a multicellular organism (systemic RNAi), has only been studied extensively in certain plants and the nematode Caenorhabditis elegans. In recent years, RNAi has become a popular reverse genetic technique for gene silencing in many organisms. Although many RNAi techniques in non-traditional model organisms rely on the systemic nature of RNAi, little has been done to analyze the parameters required to obtain a robust systemic RNAi response. The data provided here show that the concentration and length of dsRNA have profound effects on the efficacy of the RNAi response both in regard to initial efficiency and duration of the effect in Tribolium castaneum. In addition, our analyses using a series of short dsRNAs and chimeric dsRNA provide evidence that dsRNA cellular uptake (and not the RNAi response itself) is the major step affected by dsRNA size in Tribolium. We also demonstrate that competitive inhibition of dsRNA can occur when multiple dsRNAs are injected together, influencing the effectiveness of RNAi. These data provide specific information essential to the design and implementation of RNAi based studies, and may provide insight into the molecular basis of the systemic RNAi response in insects.     

Roles for Caenorhabditis elegans rad-51 in meiosis and in resistance to ionizing radiation during development

We have investigated the role of Caenorhabditis elegans RAD-51 during meiotic prophase and embryogenesis, making use of the silencing effect of RNA interference (RNAi). rad-51 RNAi leads to severe defects in chromosome morphology in diakinesis oocytes. We have explored the effect of rad-51 RNAi in mutants lacking fundamental components of the recombination machinery. If double-strand breaks are prevented by spo-11 mutation, rad-51 RNAi does not affect chromosome appearance. This is consistent with a role for RAD-51 downstream of the initiation of recombination. In the absence of MRE-11, as in the absence of SPO-11, RAD-51 depletion has no effect on the chromosomes, which appear intact, thus indicating a role for MRE-11 in DSB induction. Intriguingly, rad-51 silencing in oocytes that lack MSH-5 leads to chromosome fragmentation, a novel trait that is distinct from that seen in msh-5 mutants and in rad-51 RNAi oocytes, suggesting new potential roles for the msh-5 gene. Silencing of the rad-51 gene also causes a reduction in fecundity, which is suppressed by mutation in the DNA damage checkpoint gene rad-5, but not in the cell death effector gene ced-3. Finally, RAD-51 depletion is also seen to affect the soma, resulting in hypersensitivity to ionizing radiation in late embryogenesis.     

Duplicate genes and robustness to transient gene knock-downs in Caenorhabditis elegans

We examine robustness to mutations in the nematode worm Caenorhabditis elegans and the role of single-copy and duplicate genes in it. We do so by integrating complete genome sequence and microarray gene expression data with results from a genome-scale study using RNA interference (RNAi) to temporarily eliminate the functions of more than 16000 worm genes. We found that 89% of single-copy and 96% of duplicate genes show no detectable phenotypic effect in an RNAi knock-down experiment. We find that mutational robustness is greatest for closely related gene duplicates, large gene families and similarly expressed genes. We discuss the different causes of mutational robustness in single-copy and duplicate genes, as well as its evolutionary origin.     

Gene silencing in Caenorhabditis elegans by transitive RNA interference

When a cell is exposed to double-stranded RNA (dsRNA), mRNA from the homologous gene is selectively degraded by a process called RNA interference (RNAi). Here, we provide evidence that dsRNA is amplified in Caenorhabditis elegans to ensure a robust RNAi response. Our data suggest a model in which mRNA targeted by RNAi functions as a template for 5' to 3' synthesis of new dsRNA (termed transitive RNAi). Strikingly, the effect is nonautonomous: dsRNA targeted to a gene expressed in one cell type can lead to transitive RNAi-mediated silencing of a second gene expressed in a distinct cell type. These data suggest dsRNA synthesized in vivo can mediate systemic RNAi.     

RNAi转基因作物安全评价研究进展

在秀丽隐杆线虫中首次发现双链RNA(dsRNA)能特异性地导致基因沉默(RNAi)现象后,人们开始大量地研究RNAi技术,并将其应用于功能基因的研究,来提高作物的抗性和改良遗传育种等。本文详细介绍了RNAi的技术原理,并且对RNAi技术与传统转基因技术的区别进行分析,阐述了该技术具有重要的生物学意义,以及在农作物害虫防治领域的占据独特优势。基于RNAi技术存在的潜在脱靶效应,从改良植物、靶标生物和生态环境的3个方面具体分析该技术可能存在的风险,为RNAi技术的风险评估提供参考。由于RNAi技术仍存在风险,为了维护生态多样性和保障人们的人身安全,应尽快建立起符合实际需求的安全性评价方法,本文针对RNAi转基因作物的环境安全和食用安全2个方面的评估方案进行概述。RNAi技术对减少害虫数量、提高水稻产量、降低种植成本以及减少化学农药污染、促进农业可持续发展来说具有重要意义,但该技术仍存在风险,需要进一步监管和研究,建立完善的生态评价系统,让RNAi技术在农业生产上发挥作用。     

In vivo gene silencing in Plasmodium berghei--a mouse malaria model

RNA interference (RNAi) has emerged as a specific and efficient tool to silence gene expression in a variety of organisms and cell lines. An important prospect for RNAi technology is its possible application in the treatment of diseases using short interfering RNAs (siRNAs). However, the effect of siRNAs in adult animals and their potential to treat or prevent diseases are yet to be fully investigated. The main goal of the present study is to find out whether it was possible to carry out RNAi on circulating malaria parasite in vivo. To trigger RNAi in mouse malaria parasite, we used siRNAs corresponding to cysteine protease genes of Plasmodium berghei (berghepain-1 & 2). Intravenous injections of berghepains' siRNAs in infected animal resulted in characteristic enlargement of food vacuole in circulating parasites. Protein analysis of these treated parasites showed substantial accumulation of hemoglobin, which is reminiscent of the effect observed upon treating Plasmodium falciparum with different cysteine protease inhibitors. Parasites treated with berghepain 1 & 2 siRNAs showed marked reduction in the levels of their cognate mRNAs, thereby suggesting specific inhibition of berghepains' gene expression in vivo. We also observed the generation of approximately 25 nt RNA species from berghepains' mRNAs in the treated parasites, which is a characteristic of an RNAi phenomenon. These results thus provide evidence that beyond its value for validation of gene functions, RNAi may provide a new approach for disease therapy.     

Molecular genetics and comparative genomics reveal RNAi is not functional in malaria parasites

Techniques for targeted genetic disruption in Plasmodium, the causative agent of malaria, are currently intractable for those genes that are essential for blood stage development. The ability to use RNA interference (RNAi) to silence gene expression would provide a powerful means to gain valuable insight into the pathogenic blood stages but its functionality in Plasmodium remains controversial. Here we have used various RNA-based gene silencing approaches to test the utility of RNAi in malaria parasites and have undertaken an extensive comparative genomics search using profile hidden Markov models to clarify whether RNAi machinery exists in malaria. These investigative approaches revealed that Plasmodium lacks the enzymology required for RNAi-based ablation of gene expression and indeed no experimental evidence for RNAi was observed. In its absence, the most likely explanations for previously reported RNAi-mediated knockdown are either the general toxicity of introduced RNA (with global down-regulation of gene expression) or a specific antisense effect mechanistically distinct from RNAi, which will need systematic analysis if it is to be of use as a molecular genetic tool for malaria parasites.     

两种高效 RNA 干涉载体系统的构建及应用

在真核细胞基因功能研究中, RNA 干涉 (RNAi) 已成为一种强有力的选择性沉默基因表达的实验工具. 建立一套可在哺乳动物培养细胞中高效、经济地表达 siRNA 的载体系统是 RNA 干涉研究的必要前提之一. 从 HepG2 细胞基因组 DNA 中克隆得到 H1 全长启动子 (374 bp),以之为基础构建了两套 RNA 干涉载体系统, pSL 和带有绿色荧光蛋白 (EGFP) 标签的 pESL ,并对 p53 基因进行了相应的 RNA 干涉研究. 干涉质粒瞬时转染 HepG2 细胞后,分别利用半定量 RT-PCR 和蛋白质印迹检测 p53 表达水平. 与商品化载体 pSilencer^TM 3.1-H1 hygro 相比, pSL 和 pESL 对 p53 基因表达具有更高的干涉效率. 结果显示：干涉载体 pSL 和 pESL 能高效特异地下调目的基因表达,可作为哺乳动物中基因功能分析的有效工具.     

RNA interference for antiviral therapy

Silencing gene expression through a process known as RNA interference (RNAi) has been known in the plant world for many years. In recent years, knowledge of the prevalence of RNAi and the mechanism of gene silencing through RNAi has started to unfold. It is now believed that RNAi serves in part as an innate response against invading viral pathogens and, indeed, counter silencing mechanisms aimed at neutralizing RNAi have been found in various viral pathogens. During the past few years, it has been demonstrated that RNAi, induced by specifically designed double-stranded RNA (dsRNA) molecules, can silence gene expression of human viral pathogens both in acute and chronic viral infections. Furthermore, it is now apparent that in in vitro and in some in vivo models, the prospects for this technology in developing therapeutic applications are robust. However, many key questions and obstacles in the translation of RNAi into a potential therapeutic platform still remain, including the specificity and longevity of the silencing effect, and, most importantly, the delivery of the dsRNA that induces the system. It is expected that for the specific examples in which the delivery issue could be circumvented or resolved, RNAi may hold promise for the development of gene-specific therapeutics.